IgA nephropathy (IgAN) is the most common form of glomerulonephritis in the world and is a leading cause of end-stage renal disease. Although the mechanisms of its pathogenesis remain unclear, reports from several laboratories indicate a key role for an aberrantly glycosylated IgA1 present in the circulation and renal deposits. This IgA1 is galactose (Gal)-deficient in some of its 3-5 O-linked glycans in the hinge region (HR) of the heavy chain. Thus, in patients with IgAN, the IgA1 O-linked glycans are truncated, with terminal N acetylgalactosamine (GalNAc) or sialylated GalNAc. A variety of methods (glycan-specific lectin binding assays, MALDI-TOF MS methods, and chromatographic analysis of glycan composition) have detected clear differences between normal healthy controls'and IgAN patients'O-glycan populations in IgA1. However, the sites of attachment of normal and Gal-deficient glycans remain to be defined. Thus, a method is needed that can provide detailed structural information about the population of O-glycopeptides within each sample. Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) provides unmatched mass accuracy in the identification of biomolecules, including the population of IgA1 O-glycopeptides enzymatically released from serum IgA1. We have recently localized (for the first time by a direct method) the multiple O-glycan chains in three different IgA1 HR glycopeptides by electron capture dissociation (ECD) FT-ICR tandem MS. This methodology enables us to test our hypothesis that the populations of serum IgA1 O-glycans differ in composition and sites of attachment in patients with IgAN when compared to normal healthy and disease controls and, furthermore, that these differences can serve as markers of the disease and define the pathological features. To establish accurate profiles of IgA1 glycoforms in IgAN patients we propose the following: 1. Provide an FT-ICR accurate mass profile of serum IgA1 glycoforms found inpatients with IgAN as well as a baseline profile from normal healthy and disease controls. and 2. Localize sites of O-glycan attachment in individual serum IgA1 glycoforms from patients with IgAN, normal healthy controls, and patients with other forms of glomerulonephritis by ECD FT-ICR tandem mass spectrometry. We perform this analysis on a total of 115 samples (45 IgAN, 25 lupus nephritis, and 45 healthy controls). These studies will provide detailed structural information about the aberrant glycosylation patterns found in IgAN, give insights into the pathogenesis of this disease, and may provide a novel noninvasive method for diagnosis of IgAN. PUBLIC HEALTH RELEVANCE: This two year project seeks to use novel mass spectrometry techniques to define the sites of aberrant O-glycosylation of serum IgA1 in patients with IgA nephropathy (IgAN). This can only be done in context of understanding the sites of IgA1 O-glycosylation in normal healthy controls and disease controls. The results will provide insights into the pathology of the IgAN and may provide a non-invasive method of diagnosing th3disease.